Anti-leukemia activity of CD33-specific chimeric cytotoxic T lymphocytes is impaired by addition of the CD28 costimulatory endodomain

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T lymphocytes genetically modified to express tumor antigen-specific chimeric immune receptors (CAR) have a limited response to tumor cells in vivo due to their lack of costimulatory signals required for the full activation of resting T cells. By contrast, EBV-specific cytotoxic T cells (EBV-CTLs) expand and have long-term activity in vivo due to the sustained costimulation provided by the EBV-infected cells produced by this persistent virus. We exploited this phenomenon and redirected EBV-CTLs against CD33, a surface protein expressed on the malignant blasts of acute myeloid leukemia (AML) cells. EBV-CTLs generated from six EBV-seropositive donors were transduced using a retroviral vector encoding the CD33 specific CAR. cRCD33-EBV-CTL maintained their pre-transdcution immunophenotype and killed EBV-LCL and CD33⁺ targets (specific lysis respectively of 30% and 35% at E:T ratio 25:1). They produced Th1 and Th2-type cytokines (IFN-γ, IL-5) on exposure to CD33⁺ targets. Addition of the CD28 intracellular domain did not increase cytotoxicity to CD33⁺ targets. Preincubation of CD33⁺ cells with the CD33-blocking MoAb resulted in up to 40% inhibition of lysis and up to 60% inhibition of cytokine release by cRCD33-EBV-CTLs, whereas preincubation of CD33⁺ targets with class I and II blocking MoAb had no significant effect, confirming the specificity of the TCR interactions with CD33. Importantly, long-term culture-initiating cell assay demonstrated that gene-modified EBV-CTLs had high and long-term toxicity against CD33⁺ progenitor cells isolated from bone marrow samples. To assess activity in vivo, NOD-SCID mice bearing a human CD33⁺ AML were injected with EBV-CTLs x4 weekly starting 5 days after tumor inoculation. Significant tumor reduction by in vivo bioluminescence imaging was only observed in mice treated with the cRCD33-EBV-CTLs (p<0.05). Immunohistologic analysis showed the presence of a majority of CD8+ and some CD4⁺ human T cells in the tumors of treated mice. Incorporation of the CD28 endodomain resulted in less tumor-infiltrating T cells in mice treated with cRCD33CD28-EBV-CTLs. There was no significant difference in the chemokines receptor expression on cRCD33CD28-EBV-CTLs but their rate of apoptosis was 16 % higher (p<0.05) than the one of cRCD33-EBV-CTLs suggesting a deleterious role of CD28 signaling in this context. In summary, we show that EBV-CTLs redirected against a myeloid leukemia antigen are functional in vitro and in vivo in mice. We conclude that EBV-CTL expressing the CD33 chimeric receptor may be appropriate for further study as a therapeutic modality for the treatment of patients with advanced or refractory myeloid leukaemia.